A principal unmet need in glaucoma is that medical or surgical intraocular pressure reduction is the only clin- ically-approved treatment. But vision cannot be restored because retinal ganglion cell (RGC) loss is irre- versible. Glaucoma, the 2nd leading cause of worldwide blindness, afflicting greater than 60M, is character- ized by RGC loss, and optic nerve defects. Knowledge gaps to improved therapy include how to achieve neuroprotection, i.e., preventing RGCs from dying, or, to use cell-based therapy to re-grow or replace. Ad- ministration route, dosage, and adverse effects limit clinical application of neuroprotection and cell trans- plantation. Studying a new treatment using extracellular vesicles (EVs), biologically-active 50-150 nm di- ameter nanoparticles derived from stem cells, the proposal fills a gap and urgent need in the development of new treatments to prevent RGC death and vision loss for glaucoma patients. Their stability, biocompatibility, biological barrier permeability, and low toxicity render EVs attractive vehicles as delivery therapies to the eye. EVs represent a potential clinically applicable means to prevent RGC, axonal, and visual functional loss and decreasing the excitotoxic and inflammatory component of glaucoma. Our central hypothesis is that EVs can be designed and optimized to specifically target RGCs as a basis for precision treatment of glau- coma and, ultimately, other retinal diseases. We propose to test engineered EVs as a novel cell-free means to specifically target neuroprotection to RGCs and to fill the knowledge gaps that presently prevent clinical translation of EVs for retinal disease. Our specific aims are Aim 1: Determine the time course and factors regulating the distribution of EVs in the vitreous and retina, and optimize EV delivery to retina. Aim 2: Develop and optimize novel engineered EVs to specifically target RGCs. Successful completion of Aim 1 will optimize delivery of EVs to the retina following intravitreal injection. Aim 2 will guide administration of EVs to produce innovative, specific, targeted delivery into RGCs, allowing specificity for treatment at the major pathophysiological site of glaucoma. Fulfillment of these objectives will set the stage to develop glau- coma therapeutics using EVs by optimizing administration, and by specific RGC-targeted EVs. The study of- fers promise to save sight via development of safe, effective, and cost-efficient therapy to restore or prevent loss of sight in patients with glaucoma. This contribution is expected to be significant because these studies will provide a basis to develop EVs as a therapy for glaucoma, either primarily restoring RGC function and axonal growth, or optimizing existing therapies such as RGC transplants. Innovative features of this work are cell-free therapy of glaucoma, novel targeted EVs binding RGC-specific receptors for specific RGC ac- tion, and novel delivery materials for EVs. RO1 applications are expected to follow, to further examine mechanisms of EV actions and develop retinal cell-specific targeted, safe, low-cost therapeutics.